Sequencing-by-synthesis involves the template-dependent addition of nucleotides to a template/primer duplex. Traditional sequencing-by-synthesis is performed using dye-labeled terminators and gel electrophoresis (so-called “Sanger sequencing”). See, e.g., Sanger, F. and Coulson, A. R., 1975, J. Mol. Biol. 94: 441-448; Sanger, F. et al., 1977, Nature. 265(5596): 687-695; and Sanger, F. et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 75: 5463-5467. Recently, single molecule sequencing methods have been proposed that provide increased resolution, throughput, and speed at reduced cost. For example, a sequencing-by-synthesis method that results in sequence determination without consecutive base incorporation, has been proposed by Braslavsky, et al., Proc. Nat'l Acad. Sci., 100: 3960-3964 (2003). These methods do not rely on the user of terminator nucleotides as in Sanger sequencing. Instead, template/primer duplex is anchored directly, or indirectly (e.g., via a polymerase enzyme) to a surface and labeled nucleotides are added in a template-dependent manner.
A challenge that has arisen in single molecule sequencing involves the ability to sequence through homopolymer regions (i.e., portions of the template that contain consecutive identical nucleotides). Often the number of bases present in a homopolymer region is important from the point of view of genetic function. Many polymerase enzymes used in sequencing-by-synthesis reactions are highly-processive and tend to add bases continuously in a homopolymer region. It is often difficult to resolve the number of nucleotides in a homopolymer due to the difficulty in distinguishing between the incorporation of one or two labeled nucleotides and the incorporation of a greater number of nucleotides.
A need therefore exists for nucleotide analogs that promote accurate base-over-base incorporation in sequencing-by-synthesis reactions.